Mutations in proteins involved in post replicative DNA repair are associated with ~80% of Human Nonpolyposis Colorectal Cancer (HNPCC) occurrences. More specifically, these mutations occur in the MutS and MutL repair proteins involved in DNA mismatch repair. The long term goal of this proposal is to characterize the interactions of yeast Msh2-Msh6 (yMutS alpha), a MutS homolog, with DNA substrates containing Watson-Crick base-base mismatches and single nucleotide insertion-deletion loops (IDLs). Crystal structure data indicate MutS binds mismatched DNA into a kinked conformation, such that the DNA is slightly bent at the DNA binding domain of the protein. AFM imaging data indicate that MutS is also observed to bind DNA into a straightened and a strongly bent conformation. These observations taken together suggest that MutS utilizes a binding and bending mechanism in order to search along the DNA strand for mismatches. This binding and bending mechanism lends the system to the use of Florescence Resonance Energy Transfer (FRET) as a means to observe these binding dynamics and gain information regarding the lifetimes of each possible bent state, frequency of occurrence, and the kinetics associated with the transitions to and from each state. These experiments will be performed using through prism total internal reflectance fluorescence (TIRF) microscopy in conjunction with FRET. The energy exchange from the fluorophores on either end of a mismatch or damaged base site act as a molecular ruler providing information regarding the proximity of the dyes to one another, and therefore, the conformational states involved in the repair and/or the damage response process. Using single molecule FRET, I will compare the conformational binding dynamics of yMutS alpha on G/T mismatches, C/C mismatches, and T-bulge IDLs to those dynamics observed on homoduplex DNA. I will also be investigating the conformational dynamics observed on damaged DNA substrates (O6-methyl G/T). This damaged DNA substrate is similar in nature to damaged DNA generated by methylating agents that are active in chemotherapeutic drugs. This particular damaged substrate is associated with the initiation of DNA repair induced apoptosis. Interactions of DNA binding domain mutants with mismatched DNA and damaged DNA substrates will be characterized. By better understanding these dynamics we will be able to elucidate the mechanism by which mismatch repair on damaged DNA leads to cell death and how cells that are mismatch repair deficient are able to resist the cytoxic effects of chemotherapeutics.